The human pathogen hepatitis B virus (HBV) belongs to the family of Hepadnaviridae, a group of small, hepatotropic DNA viruses that also include closely related animal viruses, such as the duck hepatitis B virus (DHBV). Hepadnaviruses contain a small (ca. 3-kb), partially double-stranded (ds-), relaxed circular (rc) DNA genome enclosed within an icosahedral capsid that is, in turn, formed by multiple copies (240 or 180) of the viral capsid or core protein. All hepadnaviruses replicate their genomic DNA via an RNA intermediate, termed the pregenomic RNA (pgRNA), by reverse transcription. Upon entering the host cells, the virion rcDNA is released into the nucleus for conversion into a covalently closed circular (ccc) DNA, which then serves as the viral transcriptional template for the synthesis of all viral RNAs, including pgRNA, by the host RNA polymerase II. After being packaged together with the viral reverse transcriptase (RT) protein into assembling immature nucleocapsid (NC), the pgRNA is converted by the multifunctional RT, first to a single-stranded DNA (ssDNA) and then to the characteristic rcDNA. The mature (i.e., rcDNA-containing) NCs are then encapsulated by the viral envelop proteins and secreted extracellularly as virions, or they can deliver their rcDNA content to the nucleus to be converted to more cccDNA via an intracellular cccDNA amplification pathway.
The HBV core protein (HBc) consists of two separate domains: the N-terminal domain (NTD), which is sufficient to form the capsid shell, and the C-terminal domain (CTD), which is dispensable for capsid assembly but nevertheless essential for viral replication. The CTD is highly basic and dynamically phosphorylated, which is thought to be important for viral RNA packaging and DNA synthesis. The NTD has also been shown to play a role in viral DNA synthesis beyond its role in capsid assembly.
The two alternative fates of mature NCs (i.e., envelopment versus cccDNA amplification) are known to be regulated by the viral envelope proteins. Since HBc forms the NC shell, it is also likely to play a key role in these processes. On the other hand, the HBc CTD harbors the nuclear localization signal (NLS) and thus is thought to play an important role in delivering the rcDNA in mature NCs to the nucleus for cccDNA formation. Since at least partial disassembly (uncoating) of the mature NCs is required to allow rcDNA release to the host cell nucleus for cccDNA formation, NC stability or integrity likely plays a critical role in cccDNA formation. In established human hepatoma cells in culture, which have limited ability to support HBV cccDNA formation, a processed form of rcDNA called protein-free (PF) or deproteinated (dp) rcDNA, also accumulates to high levels. PF-rcDNA is derived from rcDNA, but the viral RT protein, which is used as a protein primer to initiate viral DNA synthesis and remains attached to rcDNA in mature NCs, has been removed. At least partial uncoating of the mature NCs is also thought to be required for the removal of RT from rcDNA and the generation of the PF-rcDNA. Thus, regardless of whether PF-rcDNA is a true intermediate during the conversion of RC to cccDNA, it may be a useful marker for the uncoating of mature NCs. There is an ongoing need for improved approaches that are suitable for analyzing the effect of multiple test agents on the role of HBc in coordinating the two alternative fates of mature NCs, and how or if the formation of cccDNA can be manipulated for use in prophylaxis and/or therapy of HBV. The present disclosure is pertinent to this need.